Methods and compositions for the treatment and prevention of lung disease

ABSTRACT

Methods and compositions for the treatment of lung disease such as emphysema and/or bronchopulmonary dysplasia in a mammal. Also disclosed are methods and compositions promoting the formation of alveolar septa and increasing the gas-exchange surface area of a mammalian lung, and for the prevention and/or treatment of alveolar destruction.

This application claims priority under 35 USC§ 119(e) to ProvisionalApplication No. 60/129,216 filed Apr. 14, 1999.

FIELD OF THE INVENTION

This invention concerns the use of isotype-specific retinoic acidreceptor (RAR) agonists for the inhibition of alveolar destructionand/or to promote the formation of alveoli in mammalian lung tissuedeficient in adequate numbers of functional alveoli.

BACKGROUND OF THE INVENTION

Among aerobic animals, the lung functions to provide an interface forthe exchange of gases between blood and the atmosphere. The agents ofthis exchange are numerous small sacs termed alveoli (in adult humansabout 300,000,000 per lung) that provide a gas permeable-liquidimpermeable barrier between the gas and liquid phases. Between thealveoli are numerous capillaries carrying deoxygenated blood to the lungfrom the tissues and oxygenated blood from the alveoli to the tissues.The partial pressure of oxygen in the lungs is. approximately. 100 mm Hgat sea level; at this pressure the binding of oxygen by hemoglobin inthe erythrocytes is favored. The alveoli thus provide a means forpresenting the oxygen to hemoglobin to permit the conversion ofdeoxyhemoglobin to hemoglobin. Because the exchange occurs at thesurface of the gas/blood barrier, alveoli have evolved as a means forproviding extremely high surface area in a compact overall area, thusmaximizing possible gas exchange. Lack of adequate gas exchange wouldlead to disability, which could progress to death.

Diseases that result in fewer alveoli therefore are quite serious, andare common causes of inadequate oxygenation and resultant disability anddeath. Among such diseases are bnrochiopulmonary dysplasia (BPD) andemphysema. BPD is a disease of prematurely born infants, and ischaracterized mainly by a failure of the infant to form a sufficientnumber of appropriately-sized alveoli. Emphysema, a disease of middleand advanced age, appears to be due to progressive proteinase-inducedalveolar destruction.

The process of alveoli formation is reasonably well understood from agross developmental standpoint, and seems to be similar in rat, mouse,and human, the major species studied. The process includes thesubdivision (septation) of the saccules that constitute the gas-exchangeregion of the immature lung. Septation results in the formation ofsmaller, more numerous gas-exchange structures (alveoli). The timing ofthe onset and cessation of septation vary among species, but both onsetand cessation are critical to the formation of alveoli of the size andnumber needed for adequate oxygenation.

The molecular basis of the initiation and cessation of alveoli formationare not as well understood as the structural events and timingaccompanying alveoli development. Knowledge of the molecular signalsthat initiate and end septation, and that govern the spacing of septarelative to the O₂-demand, are virtually unknown; however, several linesof evidence suggest that certain retinoids (retinoic acid and itsderivatives) may play a key signaling role. In Massaro et al., NatureMedicine 3:675 (1997), hereby incorporated by reference herein, ratswere treated with elastase, causing destruction of alveolar walls in amanner similar to that seen in emphysema. Treatment of the rats withall-trans-retinoic acid (ATRA), an agonist of all RAR isotypes, appearedto reverse this destruction. Similarly, treatment of newborn rats (whichare born with immature lungs lacking an adult complement of alveoli)with ATRA induced the formation of an increased number of alveoli inrats without enlarging the lung. See Massaro et al., Am. J. Physiol.270: L305 (1996) incorporated by reference herein.

ATRA can have a multiplicity of physiological effects. The retinoidreceptors, when bound by an appropriate ligand, are mediators of variouslife processes, including reproduction, metabolism, differentiation,hematopoiesis, and embryogenesis.

There is therefore a need for methods and compositions that provide apracticable means for inhibiting alveolar destruction and/or promotingthe formation of alveoli in a postnatal aerobic animal, particularly amammal such as a human. Additionally, there is a need for therapeuticmethods that are able to more specifically treat such a conditionwithout a high likelihood of serious side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph of a histological section of the lung of a14-day old rat injected intraperitoneally daily from age 3 to 13 dayswith cottonseed oil (control).

FIG. 1B is a photomicrograph of a histological section of the lung of a14-day old rat injected intraperitoneally daily from age 3 to 13 dayswith an RARβ agonist dissolved in cottonseed oil.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions forpromoting the formation of alveoli in mammalian lung tissue. In oneembodiment the invention comprises a therapeutic method for inducing theformation of alveoli in mammalian lung tissue by administration of acomposition comprising a therapeutically effective amount of an ligandthat is an RARγ agonist and that does not have RAR agonist activity atall RAR isotypes. In a preferred aspect of this embodiment, the RARγagonist has specific RAR modulating activity at the RARγ receptor, andis not an agonist of the RARβ receptor. In another preferred aspect, theRARγ receptor agonist has specific RAR modulating activity at the RARγreceptor and is not an agonist of the RARα receptor. In a particularlypreferred aspect of this embodiment, the RARα receptor agonist hasspecific RAR modulating activity at the RARγ receptor and is notspecific to either the RARβ or the RARβ receptor. Even more preferably,the ligand is specific to RARγ isoforms. Preferably such a ligand is aretinoid.

By “specific RAR modulating activity” is meant that such a compound hasa disassociation constant (K_(D)) (the ligand concentration at which 50%of the target RAR receptors are complexed with the ligand) at an RARreceptor at least 10 times, preferably at least 25 times, even morepreferably at least 50 times, and most preferably at least 100 timesgreater than the K_(D)for the binding of the same ligand to an RXRreceptor. Determination of the K_(D) at an RAR or RXR receptor for agiven ligand is a routine matter. Membrane preparations of host cellsexpressing a cloned RAR or RXR receptor, and the amino acid andnucleotide sequences of such receptors, has been described in variouspublications available and within the knowledge of, the person orordinary skill in the art. For example, U.S. Pat. No. 5,776,699, toKlein et al., describes assays employing RAR and RXR, and PCTPublication No. W093/11755 discloses ligand binding assays. Thesereferences are now incorporated by reference herein in their entirety.

By “agonist” is meant a retinoid receptor ligand that will cause theactivation of transcription at a gene having an appropriate retinoidreceptor response element.

By “antagonist” is meant a retinoid receptor ligand that will inhibitthe activation of transcription by the retinoid receptor at a genehaving an appropriate retinoid receptor response element in the presenceof an agonist of the retinoid receptor.

By “inverse agonist” is meant a retinoid receptor ligand that willinhibit the expression of transcription at a gene having an appropriateretinoid receptor response element beyond a basal expression levelexisting in the absence of an agonist of the retinoid receptor.

In another embodiment, the invention is directed to therapeuticcompositions for the treatment of an emphysemic mammal, or of a mammalsuffering from bronchopulmonary dysplasia, comprising a therapeuticallyeffective amount of an ligand that is an RARγ agonist and that does nothave RAR agonist activity at all RAR isotypes. In a preferred aspect ofthis embodiment, the RARγ agonist has specific RAR modulating activityat the RARγ receptor, and is not an agonist of the RARβ receptor. Inanother preferred aspect, the RARγ receptor agonist has specific RARmodulating activity at the RARγ receptor and is not an agonist of theRARα receptor. In a particularly preferred aspect of this embodiment,the RARα receptor agonist has specific RAR modulating activity at theRARγ receptor and is not specific to either the RARβ or the RARαreceptor. Even more preferably, the ligand is specific to RARγ isoforms.Preferably such a ligand is a retinoid.

It is contemplated that the RARγ agonist may be used either as the onlyactive ingredient in the composition, or in combination with one or moreadditional therapeutically active ingredient. In one aspect, theadditional therapeutically active ingredient is a retinoid; in apreferred aspect, an additional therapeutically active ingredient isanother RAR-active ligand, for example, all-trans-retinoic acid.

By “RAR-active retinoid” is meant that the retinoid has agonist,inhibitory, or inverse agonist (negative hormone) activity at an RARreceptor.

By “therapeutically effective amount” is meant that the amount of theRAR-specific therapeutic agent is sufficient, either as the result of asingle dose, or as the result of multiple doses over the term oftherapy, to decrease the rate of alveolar destruction in an emphysemicmammal, or to promote the growth of alveolar septa in said mammal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods for thetreatment or prevention of alveolar destruction and/or to promote theformation of alveoli is mammalian lungs deficient in adequate numbers offunctional alveoli. Such methods and compositions involve RAR-activeretinoids, therapeutic compositions containing such agents, and methodsfor their use.

The retinoid receptors are part of the steroid/thyroid/vitamin Dsuperfamily of nuclear receptors. The retinoid receptors include theretinoic acid receptors (RAR) and the retinoid X receptor (RXR). The RARand RXR receptors are single chain polypeptides containing a number ofstructural domains in common: a ligand binding domain, asequence-specific DNA binding domain, and a leucine zipper motif. In thepresence of ligand, the single RAR or RXR chains can, by virtue of theleucine zipper, form dimers. The RAR chain is believed to exist in vivoexclusively as an RAR/RXR heterodimer. RXR may form heterodimers withRAR or other members of the superfamily, such as the vitamin D receptorand the thyroid receptor.

Retinoid receptor diners are effective transcription factors regulatinggene transcription by binding to retinoic acid response elements (RAREs)or retinoid X response elements (RXREs) present in (or near) thepromoters of retinoid responsive genes, or by negatively regulating theenhancer functions of other transcription factors.

Described RAR isotypes include RARβ, RARα, and RARγ, and described RXRisotypes include RARβ, RARα, and RARγ. Within each receptor class, theseisotypes have sequence homology, but are encoded by different genes.Within each isotype several isoforms have been described; these isoformsdiffer in their N terminals and are generated by alternative splicingand/or differential usage of more than one promoter. See e.g., Nagpal &Chandraratna, Current Pharm. Design 2:295-316 (1996) and Mangelsdorf etal., The Retinoid Receptors in The Retinoids: Biology, Chemistry andMedicine Ch. 8(2d ed. Sporn et al. eds. 1994), both of which are herebyincorporated by reference herein.

Depending both upon the ligand and the nature (e.g., RAR or RXR;isotype; isoform) of the monomer chains contained in the dimers, anenormous variety of biological responses regulated by the retinoidreceptors are possible.

Ligands specific to RAR (e.g., ATRA) or RXR (e.g., TTNB), and tospecific RAR isotypes have been described. See e.g., Nagpal &Chandraratna, Current Pharm. Design 2:295-316 (1996), incorporated byreference herein. Therefore, the design and/or selection of RAR-specificligands, and of RAR isotype-specific ligands is well within the abilityof the person of ordinary skill in the art.

By “specific” to a given retinoid receptor is meant that thedisassociation constant (K_(D)) for the binding of the ligand to a giventarget receptor or receptor isotype or isoform is at least 10 timeslower than the K_(D) value for the ligand and a non-target receptor orreceptor isotype or isoform. K_(D) is defined as the concentration ofligand at which 50% of the receptors are ligand bound. Even morepreferably, K_(D) is at least 25 times lower for the target receptorthan for untargeted receptors. Most preferably, K_(D) is at least 50, orat least 100, times lower for the target receptor than for untargetedreceptors.

An aspect of the present invention comprises compositions for thetreatment or prevention of alveolar destruction and/or the promotion ofalveolar formation in a mammal. Such compositions comprise a ligand thatis an RARγ agonist and that does not have RAR agonist activity at allRAR isotypes. In a preferred aspect of this embodiment, the RARγ agonisthas specific RAR modulating activity at the RARγ receptor, and is not anagonist of the RARβ receptor. In another preferred aspect, the RARγreceptor agonist has specific RAR modulating activity at the RARγreceptor and is not an agonist of the RARα receptor. In a particularlypreferred aspect of this embodiment, the RARα receptor agonist hasspecific RAR modulating activity at the RARγ receptor and is notspecific to either the RARβ or the RARα receptor. Even more preferably,the ligand is specific to RARγ isoforms. Preferably such a ligand is aretinoid.

Alveolar destruction may be the result of a pathological condition suchas emphysema. Treatment to promote alveolar formation may be in responseto a condition such as bronchopulmonary dysplasia (BPD).

Another aspect of the invention is methods for the treatment oftreatment or prevention of alveolar destruction and/or the promotion ofalveolar formation in a mammal, comprising administering a therapeuticamount of a composition comprising a retinoid receptor ligand that is anRARγ agonist and that does not have RAR agonist activity at all RARisotypes. In a preferred aspect of this embodiment, the RARγ agonist hasspecific RAR modulating activity at the RARγ receptor, and is not anagonist of the RARβ receptor.

In another preferred aspect, the RARγ receptor agonist has specific RARmodulating activity at the RARγ receptor and is not an agonist of theRARβ receptor. In a particularly preferred aspect of this embodiment,the RARα receptor agonist has specific RAR modulating activity at theRARγ receptor and is not specific to either the RARβ or the RARαreceptor. Even more preferably, the ligand is specific to RARγ isoforms.Preferably such a ligand is a retinoid.

The above-mentioned embodiments and aspects are clearly useful for thetreatment of medical conditions, such as emphysema and BPD, which havebeen heretofore difficult or impossible to treat without a lungtransplant. As indicated above, use of the RARβ agonist in combinationwith another agent with activity in treatment of alveolar deficiencies,such as all-trans-retinoic acid, is useful as well. Such methods andcompositions may provide a synergistic therapeutic effect and/or permitthe use of lower effective doses of ATRA or another therapeutic agent(and the RARβ agonist), thus reducing the prospect of undesired sideeffects resulting from the use higher concentrations of either agentwhen used alone.

The compositions of the present invention can be administered in anytherapeutically effective manner or form, and in conjunction with anypharmacologically effective vehicle. For example, in a particularlypreferred aspect, the compositions of the invention may be administeredin the form of an inhalant as a powdered or liquid aerosol. Such aformulation may comprise the active agent solubilized in a micronizedhydrophobic/hydrophilic emulsion. Such compositions are well known tothose of skill in the art.

Alternatively, the compositions may be administered systemically, suchas intravenously by infusion, or by intraperitoneal injection. Forintravenous administration, the necessary composition ofpharmacologically effective infusion solutions, such as the properelectrolyte balance and tonicity are well known to those of skill in theart, and therefore formulation of the compositions of the presentinvention with such solutions would be well within the ability of such aperson with the disclosure of this application. Similarly,administration of drugs by intraperitoneal injection is well known, andpharmacological vehicles are well known.

Synthesis of candidate compounds having specific RAR modulating activityis well-known in the art. For example, among other references, thesynthesis of RAR ligands is disclosed in commonly owned U.S. Pat. Nos.5,739,338; 5,728,846; 5,760,276; 5,877,207; the disclosures of all ofwhich is incorporated by reference herein. The synthesis ofRARγ-specific agonists is disclosed in Swann et al., European PatentPublication No. EP 0 747 347 and in Reczek et al., Skin Pharmacol. 8:292-299 (1995), also incorporated by reference herein. Additionally, theconstruction of combinatorial libraries of compounds suitable forscreening as RAR-selective ligands is now commonplace and well known tothose of skill in the art.

Likewise, methods of screening candidate compounds for specific RARmodulating activity is routine and well-known in the art. For example,U.S. Pat. No. 5,455,265, incorporated by reference herein, describes achimeric receptor transactivation assay which tests for RAR-agonistactivity in the RAR-α, RAR-β, RAR-γ, RXR-α receptor subtypes. Briefly,this assay employs chimeric proteins containing an RAR ligand-bindingdomain and a heterologous polypeptide segment having the ability to bindto a response element (RE), in turn facilitating transcription of aspecific, measurable target gene, such as chloramphenicolacetyltransferase (CAT). Only an agonist of the given RAR isotype whoseligand binding domain comprises part of the chimeric protein will permitthe activation of CAT transcription and expression. These results can becompared with those obtained using chimeric proteins having non-targetligand binding moieties. For example, those ligands able to stimulateCAT synthesis when used in combination with chimeric proteins having RARbinding domains, but not those having RXR ligand binding domains, willbe RAR specific agonists.

variation of this transactivation assay permits testing ligands as RARantagonists, or antagonists of a given target isotype. A competitiveassay involving the use of a stimulatory concentration of a knownagonist of a given receptor (for example, ATRA is known to be an agonistof all RAR isotypes), and measuring the reduction in a reporter geneexpression (e.g., CAT expression) as a function of the added candidatecompound.

Additionally, straightforward receptor binding studies can be performedas competition, rather than transactivation assays. For example, andwithout limitation, membrane preparations containing cloned retinoidreceptors can be used, and the receptors loaded with a knownradiolabeled ligand. The release of radioactivity from thesepreparations as a function of test compound concentration can bedetermined. The lower. the Kd for a given ligand, the more likely theligand will be effective as a modulator of receptor activity. Furtherdisclosure is available in, e.g., U.S. Pat. No. 5,776,699, previouslyincorporated by reference herein.

EXAMPLE 1 Involvement of RAR Receptors in Alveolus Formation

In most mammals new alveoli are formed postnatally; thus the lung. ofmany newborn mammals is immature, and not merely. a smaller version ofthe adult lung. For example, in humans alveolus formation can continueup to the age of 20.

The study of alveolus development in newborn mammals, in this casenewborn rats, therefore provides an opportunity to study the effect ofvarious agents on alveolus formation. The rate of increase in specificlung volume (expressed as cm³/100 g body weight) in rats is greatestwithin the first 10 days following birth, after which it increases at aless steep rate.

However, the lung's efficiency is determined not merely by the volume ofair that can be contained in the lung, but by the alveolar surface area,which is a function of tissue growth within the lung. Burri et al.,Anat. Rec. 178:711-730 (1973) and Burri, Anat. Rec. 180: 77-98 (1974)performed extensive studies of the postnatal rat lung and demonstratedthat three developmental phases can be shown. In the first stage (day1-4) there is a massive expansion of the lung's volume. In the secondstage, there is an increase in lung tissue proliferation due toseptation, including alveolar and capillary surface areas. In the finalstage (day 21 to adult) there is a period of concomitant tissueredistribution, lengthening of the septa and alveolar growth.

In this example, healthy 3-day old Sprague-Dawley rat pups were injectedintraperitonally with either cottonseed oil (carrier) or with cottonseedoil containing 1 μl/gram of a retinoid ligand specific to RAR receptorsand not to RXR receptors at a dosage of 500 micrograms per kg. Thisretinoid, termed AGN 193174, has a structure defined by the formula

and has RARβ agonist activity and RARβ and RARγ antagonist activity.Therefore, with regard to RARγ, this drug has an activity opposite tothat of ATRA.

Following the initial injection, the rat pups were subjected toidentical daily injections until day 13.A set of rat pups weresacrificed at day 4 and at day 21 by anesthesia with phenobarbitolsodium and scission of the abdominal aorta. Following sacrifice, ratlungs were fixed and histological sections prepared essentially asdescribed in Massaro et al., Am. J. Physiol. 270: L305 (1996),incorporated by reference herein. The histological sections were viewedand photographs taken under light microscopy.

The histological sections, replicated in FIGS. 1A and 1B, show that atage 14 days alveoli were larger, and the degree of sepatation less, inrats that had been treated with a RARβ agonist daily from age 3 through13 days (the sepatation stage of lung development), than in rats treatedwith cottonseed oil alone.

The differences in alveolar dimensions between =treated and untreatedrat pups were so pronounced, they were quantitated by measuring thedistance between alveolar walls (Lm) and the surface-to-volume ratio(S/V) of the gas-exchange structures (septa). The Lm in cottonseedoil-treated rats was 80 μm±2.8 μm (N=4) compared to 110 μm±7.3 μm (N=3)in RARβ agonist-treated rats (N=3; P=0.03 by Mann Whitney nonparametricanalysis). The S/V was 504 μm⁻¹±18 μm⁻¹(N=4) in cottonseed oil-treatedrats compared to 365 μm⁻¹±23 μm⁻¹(N=3) in RARβ agonist-treated rats(P=0.03). Lung volume was the same in both rat groups but surface areawas larger (634 cm²±9 cm²) in cottonseed oil-treated rats than in RARβagonist-treated rats (492 cm²±28cm², P=0.03).

These data demonstrate that treatment of immature rats with a RARβagonist (and RARβ antagonist) inhibits septation of the saccules of thegas-exchange region, resulting in larger but fewer alveoli and a lowersurface to volume ratio, and clearly suggest that antagonism of the RARαreceptor (or the RARα receptor and the RARγ receptor), but not the RARβreceptor, results in the down-regulation of naturally occurring,spontaneous alveolus formation. A clear implication of this finding isthe inverse is also true: that agonist activity at the RARα receptor,alone or in conjunction with stimulation of the RARγ receptor, isrequired for adequate sepation to occur. Thus, therapeutic treatment ofmammals with an isotype-selective RAR agonist will promote the growth ofsepta.

It is quite surprising, in light of the previous finding that ATRA (anRAR-specific agonist of RARβ, RARα, and RARγ) stimulates septation, thatan antagonist of RARγ, but not of RARβ, is able to repress septation,and that stimulation of RARβ through the action of an RARβ agonist isnot necessary for septation to occur.

These data therefore provide evidence that alveolus formation ispromoted in vivo by stimulation of RARγ (or RARα and RARγ), and thatstimulation of the RARβ receptor is not required.

The data also indicate that the increase in lung air volume seen in thefirst few days is an event independent from the presence or absence ofRAR receptors or the presence or absence of an RAR modulating ligand.The septation stage of lung development can therefore be uncoupled fromthe increase in lung volume seen immediately post birth. Thus,modulating of RAR receptors can be used to specifically affect septationwithout modulating other events in alveolar formation.

Thus, signal transduction through RAR receptors appears to be anendogenous modulator of alveolus formation. The generation of agentshaving specific RAR modulating activity (i.e., RARγ agonist activity)which do not affect the RARβ receptor permits more specific targeting ofalveolar formation, without undesired effects caused by unnecessary RARβsignaling. In addition, treatment with a RARγ agonist plus all-transretinoic acid may well augment the effect of either agent alone,allowing the use of lower doses of these drugs.

It will be recognized that the foregoing examples and preferredembodiments are exemplary only, and that the invention is defined solelyby the claims that conclude this specification.

What is claimed is:
 1. A method for the treatment or prevention ofalveolar destruction in a mammal in need thereof comprising the step ofadministering to said mammal a therapeutically effective amount of anRAR-specific RARγ agonist, wherein said RARα agonist is not an agonistof RARβ.
 2. The method of claim 1 wherein said RARγ agonist is notspecific to RARβ.
 3. The method of claim 1 wherein said RARγ agonist isnot specific to RARα.
 4. The method of claim 1 wherein said RARγ agonistis not specific to RARβ or RARα.
 5. The method of claim 1, wherein saidcomposition is administered in the form of an inhalant.
 6. The method ofclaim 5 wherein said RARγ agonist is not specific to RARβ.
 7. The methodof claim 5 wherein said RARγ agonist is not specific to RARα.
 8. Themethod of claim 5 wherein said RARγ agonist is not specific to RARβ orRARα.